The detection of specific nucleic acid sequences is commonplace in the research laboratory. Southern (J. Mol. Biol. 1975.98:503-527) teaches detection of specific sequences among DNA fragments separated by gel electrophoresis using “blotting” or transfer of the DNA fragments to a membrane, followed by hybridization of denatured DNA fragments with radioactive probes and autoradiography. This procedure has been extended to the detection of RNA molecules extracted from cells or tissues (“Northern” blotting). Further improvements have involved faster and more quantitative “dot-blotting” procedures to detect DNA or RNA from tissues or cells.
Other methods are used to detect and characterize specific nucleic acid sequences and sequence changes. These methods must be able to create detectable signals from a very low copy number of the sequence of interest. Nucleic acids detection methods include capillary gel electrophoresis (CGE) (U.S. Pat. No. 5,420,265) and signal amplification technology such as the polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202) and ligase chain reaction (LCR) (Barany, Proc. Natl. Acad. Sci. 1991.88:189).
Recently, considerable interest has been generated in the development of synthetic antisense oligonucleotides as therapeutic agents. These antisense molecules and strategies for their use have been reviewed by Agarwal (Trends in Biotechnology 1991.10:152-158) and Crooke (Antisense Research and Applications, Chapter 1, Basic Principles of Antisense Therapeutics, Springer-Verlag Press, New York, 1998). For an antisense therapeutic to be effective, the oligonucleotide must be administered to a patient and must reach the specific nucleic acid target for which it was designed. Consequently, there is a need to be able to detect oligonucleotides in bodily fluids and extracts. In animal models, radiolabeled oligonucleotides have been administered to the subject and the distribution of the oligonucleotides within the body has been assessed by extraction of the oligonucleotides followed by autoradiography (Agarwal et al. Proc. Natl. Acad. Sci. 1991.88:7595-7599). A common aspect of current procedures is the detection of large DNA or RNA molecules (>100 bp). Impraim et al. (U.S. Pat. No. 6,228,578) describe a non-radioactive hybridization assay and kit for detection of genetic defects, microbial infections or viral infections by detecting large pieces of nucleic acid molecules. The method has several prescribed steps, the first being hydrolyzing the RNA in the sample and denaturing the target DNA that is to be detected. Next, the target DNA sequence is hybridized to a complementary RNA probe to form a double-stranded DNA/RNA hybrid, which is followed by capture of the hybrid onto a solid phase where an anti-hybrid antibody has been immobilized. The non-hybridized probe is eliminated by digestion with Rnase and the bound hybrid is then detected. This method does not, however, allow for detection of small molecules such as antisense compounds 20 to 30 nucleobases in length but instead is for detection of nucleic acid molecules from organisms such as human papilloma virus and hepatitis B virus. Further, because the method is designed for detection of much larger nucleic acid molecules, its sensitivity is limited; this is because the molar sensitivity of a method is dependent on the molecular weight of the entities being detected.
Thus, the small size (20-30 bp) of oligonucleotides used for antisense therapeutics presents unique concerns for design of detection methods, such as, for example, nonspecific binding or the absence of binding to probes producing false negatives/positives. Temsamani et al. (1993) disclose a method for quantitation and detection of phosphorothioate modified oligonucleotides. In this method, oligonucleotides are first extracted from tissues and body fluids and then the first step is explicitly stated to be immobilization of an oligonucleotide to a solid support, specifically a nylon membrane. The membrane-bound oligonucleotide is then hybridized with a radioactive-labeled complementary oligonucleotide and exposed to x-ray film, or alternatively, hybridized with a probe that is chemiluminescent, either method of hybridization allowing for detection of the bound oligonucleotide. This method, however, does not employ any method for removal of non-hybridized probe, such as with a nuclease, a step that would enhance the sensitivity of the assay by decreasing the amount of noise. In fact, the detection of oligonucleotides using this assay is reported to be in the range of only as low as 1.5 nanograms
There is a need for more sensitive methods for detecting administered modified oligonucleotide compounds, such as antisense therapeutics, methods with sensitivity below the nanogram range. Highly sensitive methods would be useful for determining the concentrations of modified oligonucleotide therapeutics in animal models and/or in the clinic. Further uses would be to study the pharmacokinetic properties of oligonucleotide therapeutics in animal models and/or in the clinic.